This invention relates to electrophoretic separations of chemical species in free solution. More particularly, this invention is directed to an electrophoretic apparatus wherein a liquid support medium in a separation chamber is stabilized against thermal/density gradient induced convective flow by a Taylor vortex regime.
Electrophoretic techniques, such as electrophoresis and isoelectric focusing, have been widely used in modern biotechnology. They are recognized by skilled practitioners as powerful techniques for separation and analysis of proteins and nucleotides, particularly, but also other charged chemical species. The principle of electrophoretic separation is that molecules of different charge and/or size acquire different velocity with respect to a support medium in the presence of an electrical field. A mixture, of various charged molecules will, under the influence of an electrical field, gradually be separated into zones of concentrations of molecules possessing identical migration velocities. In the isoelectric focusing mode, the support medium is prepared to have a pH gradient across the electrical field. The charge on amphoteric molecules in the carrier medium varies with their position in the pH gradient. The molecules move toward the anode or cathode (depending on their charge) until they reach a pH zone within the support medium pH gradient where their net charge is zero--their isoelectric point. Thus molecules having like isoelectric points are focused, i.e. concentrated into the pH regions corresponding to their respective isoelectric points.
One of the principal problems experienced by early users of electrophoretic separation techniques is that associated with convective disturbances in the electrophoretic support medium. The passage of electric current through the electrophoretic medium results in Joule heating of the medium. This heat is dissipated only through the boundaries of the electrophoresis chamber, and a natural consequence is the evolution of temperature, potential and concentration gradients within the medium. Because the density of the medium is a function of temperature, potential and concentration, gradients are established, leading to convective flows. These flows readily disrupt the separation process by mixing of otherwise discrete zones in the medium.
One of the most commonly used techniques to suppress convective mixing in electrophoretic media is the use of so-called anti-convective stabilizers such as coherent or granular gels. The former are most commonly polyacrylamide or agarose and the latter is usually Sephadex. Gels have been used in the preparative applications of each of the three common electrophoretic modes, isoelectric focusing, isotachophoresis, and zone electrophoresis. There are, however, several fundamental problems associated with gel stabilized electrophoretic methods, especially in larger scale preparative applications. The manipulation and preparation of the gel based supports is time consuming and tedious. There is often a problem with adherence of the sample components to the gel which not only results in a decreased recovery but can also cause electroosmosis which has deleterious effects on resolution. In addition, the matrix must be removed from the recovered sample, and cannot generally be reused. Thus, while gel based stabilization methods are adequate for most research laboratory requirements, the upper limit of sample size is wholly inadequate for large scale separations. For that reason and because use of gels introduce complicating factors such as adsorption of analyte to the stabilizer, molecular sieving effects, or tortuosity of migration paths, there has been a significant effort in the art directed toward development of methods and equipment for electrophoretic separations in free solution.
One of the earliest methods to stabilize liquid media against convection was through use of vertical density gradients. A review of the literature will show that practitioners have also attempted to make use of temperature gradients, complicated flow methods, and hydrodynamic pumping. One of the most effective methods, in terms of sample capacity, for large scale electrophoretic methods including electrophoresis and isoelectric focusing, utilizes membranes to define subcompartments in an electrolyzer. The membranes prevent bulk flow between adjacent compartments while allowing free migration of separation substrates. Most recently, Bier and co-workers have combined compartmentalized columns and slow cylinder rotation in a device which has been commercialized as the "Rotofor".
The use of rotation of an electrophoretic separation chamber to stabilize a liquid support medium was used early by Hjerten who carried out zone electrophoresis and isoelectric focusing in a horizontal cylinder having an internal diameter of 3 mm, which is rotated (40 rpm) about the electrophoretic axis. The rotation acts to re-suspend zones of separation substrate which would otherwise sediment due to their higher density. The technique is micro-preparative at best. If the diameter of the cylinder is less than 0.8 mm, no rotation is necessary. The fluid in such a small cylinder is stabilized by its own viscosity and capillary action. Electrophoresis techniques utilizing such small diameter cylinders is referred to in the literature as capillary zone electrophoresis.
Another approach to electrophoretic separations utilizing rotational stabilization of a liquid carrier medium is the "Biostream" separator developed at the Harwell Atomic Energy Institute utilizing a concept originated by Philpot and developed by Thompson. Separation takes place in an annulus between two vertically oriented concentric cylinders. The carrier/support buffer is pumped into the chamber from the bottom. At the top is a stack of "maze plates" that divide the fluid into multiple fractions which in cross-section are concentric rings. Fluid stabilization is achieved by rotation of the outer cylinder at 150 rpm, creating a velocity gradient radially across the annulus, which maintains a laminar flow profile. The walls of the rotor and stator which define the annular space are semi-permeable and isolate electrode chambers from the separation chamber.
Because electrophoretic methods conducted in free solution offers significant functional and practical advantages over systems utilizing anti-convective stabilizers, especially in the area of preparative/industrial scale operation, there is a continuing need to develop alternative methodologies for anti-convective stabilization of liquid media under electrophoretic conditions.
Accordingly, it is an object of this invention to provide an apparatus for conducting electrophoretic separations in free fluids utilizing the phenomena of Taylor vortex flow for fluid stabilization against convective mixing.
It is a further object of this invention to provide a method for utilizing the convective circulation of the Taylor vortex to stabilize free liquid electrophoresis media against thermal, concentration and potential transients.
It is still another object of this invention to provide a construction for an apparatus for the performance of electrophoretic separations which can be scaled up directly from analytical (microliter) to industrial (multiliter) scale.
A separation apparatus usable for electrophoretic separations, including particularly zone electrophoresis and isoelectric focusing, in free liquids is provided. The apparatus is constructed to have a substantially uninterrupted annular chamber adapted to contain a support fluid and separation substrates capable of migrating relative to said fluid under the influence of an electrical field. The annular chamber is defined by walls of an outer cylinder and a substantially coaxial, rotatably mounted inner cylinder. Preferably at least one of the cylinders is a right circular cylinder. The inner cylinder can be in the form of a solid cylinder or a tube having an outer diameter less than the inner diameter of the outer cylinder at respective axially aligned locii.
The annular chamber is provided with means for applying an electrical field between axially remote anodic and cathodic regions of the chamber. The electric field is preferably applied through electrodes in current conducting communication with the respective anodic and cathodic regions of the annular chamber. The electrodes can be in direct contact with a support fluid in the chamber or, preferably, each electrode is positioned in an electrolyte, itself in current conducting communication with the support fluid. The electrolyte in contact with each electrode can be separated from the support fluid by an electrical current transmitting membrane structure, such as a porous glass membrane or a semi-permeable membrane.
The separation apparatus also includes a means for rotating the inner cylinder relative to the outer cylinder at a rotational frequency (F) where F is at least as high as the critical frequency (F.sub.c) for the onset of Taylor vortex flow in the annular chamber. The critical frequency for any given apparatus can be calculated from the formula provided hereinbelow. The critical Taylor vortex frequency is unique to each separation apparatus dependent on specific physical parameters unique to that apparatus and the viscosity of the support fluid in the annular chamber. The inner cylinder is preferably rotated utilizing a variable speed stepping motor. The preferred rotational frequency of the inner cylinder during use of the separation apparatus is F.sub.c .ltoreq.F&lt;10F.sub.c. Higher rotational frequencies can be utilized, but without known advantage.
The present separation apparatus is also equipped with means for separating fluid in axially discrete portions of the annular chamber from fluid in other axially discrete portions of the annular chamber. In a preferred embodiment the fluid separation means includes a valved elution port communicating with the separation chamber and positioned so that at least a portion of the fluid can be eluted from the annular chamber through the port by gravity flow when the chamber is in a vertical orientation. Preferably the valved outlet is located at or near an axial end of the annular chamber and thus proximal to the lowest point of the annular chamber when situated in a vertical orientation.
Alternatively, the means for separating axially discrete portions of fluid in the chamber includes a port located proximal to an axial end of the annular chamber which is the uppermost end of the chamber in vertical orientation. Axially defined portions of the fluid in the annular chamber can be removed, for example by a syringe inserted through said port and used to withdraw axially discrete portion of the fluid in the chamber. The upper port can optionally serve also as a vent for gases formed at the electrodes during apparatus operation when the electrodes are positioned in the chamber.
In a preferred embodiment the anodic region and cathodic region of the annular chamber are each located proximal to a respective opposite axial ends of the annular chamber so that the electric field spans substantially the entire axial length of the chamber.
Optionally the separation apparatus of this invention can include means for controlling temperature of the fluid contained in the annular chamber. Thus in a simple embodiment the temperature control means can include a coolant jacket in thermal communication with a wall of the chamber. Liquid of predetermined temperature can be circulated through the coolant jacket, for example, to minimize temperature increase of the support fluid during performance of electrophoretic separation in the apparatus. Temperature control can be especially important when the separation substrates are known to be thermally labile.
The separation apparatus can also be equipped with a means for detecting the axial locii of discrete concentrations of separation substrates in the support fluid. Thus, for example, the apparatus can be designed to accommodate a traveling light source and detector which can be moved axially along the length of the annular chamber to detect axially defined portions of support fluid which contain concentrations of separation substrates. Where such a detection system is used, it is preferred that the outer cylinder be formed at least partially from an optically transparent material, preferably quartz glass, to allow transmission of a broad range of UV and visible radiation.
Alternatively, a detection means for substrate concentrations can be utilized to analyze the support fluid as it is eluted from the separation chamber through the valved elution port. Thus the valved port can be equipped with a flow cell which includes a sensor capable of measuring, for example, pH, conductivity, or optical properties of fluid flowing from the separation chamber and through said flow cell. The signal from the sensor in said flow cell can be displayed graphically or utilized to trigger a fraction collector pre-programed to collect effluent volumes exhibiting sensed substrate concentrations above predetermined threshold values. The apparatus can be utilized in an isoelectric focusing mode wherein pH gradient-generating ampholytes are combined with the support fluid to establish a predetermined pH gradient between the anodic and cathodic regions of the annular chamber. In that mode of operation a compound of known isoelectric point can be isolated from support fluid fractions by collecting those fluid volumes having a pH corresponding to the compound's isoelectric point. Such volumes can be detected utilizing a pH sensing flow cell mounted on the elution port.
The Taylor vortex flow stabilized separation apparatus in accordance with this invention, is operated utilizing general operating procedures and techniques which have been utilized for electrophoretic separations in free fluid electrophoretic separation devices in the prior art. Thus positioning of electrodes, loading of samples, selection of support fluid composition (for example, buffer strength, buffer components, pH), selection of support fluid additives (for example, ampholytes, surfactants/surfactant micelles, urea, non-ionic densiometric agents, and the like) can be selected and utilized based on the same criteria for their selection and use in art-recognized separation equipment for electrophoretic separations in free fluid.
The present separation apparatus can be utilized to effect electrophoretic separation of a wide variety of chemical species ranging from metal ion species to amino acids, proteins, nucleotides and polynucleotides, viruses, bacteria and other whole cells. The significance of such broad application of the performance of electrophoretic methods utilizing the present apparatus is further highlighted by the fact that the Taylor vortex stabilization phenomena is such that the apparatus can be readily constructed to commercial scale specifications without loss of function or efficiency of operation.